Secretion of granules from cultured RBL-2H3 mast cells, whether induced by antigen or G protein-dependent mast cell secretagogues such as compound 48/80 or adenosine, is dependent on calcium and protein kinase C. The secretory response to all stimulants is enhanced by prior treatment of cells with cholera toxin and this enhancement is associated with increased activation of phospholipase(PL)D. PLD, rather than PLC, is primarily responsible for the generation of diglycerides and the subsequent activation of protein kinase C (see previous reports in this series). We have now examined the activation of PLD and its enhancement by cholera toxin in more detail. Our results suggest that compound 48/80 and adenosine analogs activate PLD through release of beta/gamma subunits from Gi-3 in the plasma membrane and cholera toxin facilitates this activation through release of G-beta/gamma subunits from Gs on the basis of studies with antibodies in permeabilized cells and overexpression of beta/gamma subunits in a Semliki viral expression system. Over expression of beta/gamma subunits, for example, not only enhances basal PLD activity but also enhances activation of PLD by all stimulants including calcium in permeabilized cells. On this basis we have hypothesized that beta/gamma subunits act synergistically with other signals to stimulate PLD through a recently identified pleckstrin homology (PH) domain in PLD. Overexpression of the putative PH peptide blocks activation of PLD but the specificity this action is still to be determined. Our results indicate for the first time that PLD is directly linked to receptors via trimeric GTP-binding proteins and point to a potential mechanism for the pathological effects of cholera toxin in addition to its well established actions via adenylyl cyclase. - RBL-2H3 mast cells; secretion; antigen; compound 48/80; phospholipase D; GTP-binding proteins